An RNA Guide to Maturity

Figure 1. The central dogma: DNA replication, transcription 

to RNA, then translation into protein. 

https://genius.com/Biology-genius-the-central-dogma-annotated

Every cell in our bodies contains DNA that encodes all the information that cells needs to function. Through transcription, the DNA instructions are copied into different types of RNA(Figure 1). Both DNA and RNA are made of nucleotides. There are four distinct nucleotides that base pair with each other to form the structure of the double-stranded DNA or RNA. These nucleotides are distinguishable due to differences in the nitrogenous base portion of each nucleotide. This nitrogenous base portion is responsible for the pairing, and when hydrogen bonds are formed between bases, the structure of the nucleic acid comes together.

Figure 2. Base pairs in DNA: A pairs with T, C pairs with G.

https://en.wikipedia.org/wiki/Complementarity_(molecular_biology)

The bases of DNA are adenine (A), cytosine (C), guanine (G), and thymine (T), as shown in Figure 2. A, C, and G also make up RNA, but thymine doesn't appear in RNA; it's replaced by a different base named uracil (U). C and G always base pair with one another, while A and T (U in RNA) pair. This complementary pairing only works because of the specific shape and chemical groups on each unique base. Figure 3 gives a (funny) example of two bases that cannot base pair, since their structures don't match up. It's helpful to think about puzzle pieces when conceptualizing this.

Once RNA is transcribed from its template DNA, it exits the nucleus. Some types of RNA go on to perform a cellular function themselves. Messenger RNA (mRNA) is one type of RNA that does not function as an RNA itself, but instead relays DNA's instructions to another piece of cellular machinery, the ribosome. The ribosome then uses those instructions to make proteins. Another type of RNA transcribed from DNA is called ribosomal RNA (rRNA). This type of RNA goes on to complex with proteins to form the ribosome. Just after transcription, the nucleic acid is called pre-rRNA. Before rRNA can mature and function in translation, it needs to be processed by the cell, as do many types of RNA. 

Figure 3. Nitrogenous base pair mismatch. Remember, A pairs with
T(U), G pairs with C, both with high fidelity. http://www.clontech.com/

When one of the four bases mentioned above is chemically altered during a pre-RNA's maturation process, it's called a "modified" base. One modification to the uridine nucleotide in RNA transforms it into a "pseudouridine". This was the first RNA base modification to be discovered, and is the most abundant of the many RNA modifications known today! There is evidence that, when uracil is modified in this way, it increases the rigidity of the RNA's structure, particularly its sugar-phosphate backbone. This modification increases the stability of the pre-RNA, helping it toward maturation.

Figure 4. Uridine base modification, adapted from Charette & Gray (2000)

The transformation from uridine to pseudouridine occurs via a 180º rotation of the nitrogenous base, altering the bond that connects the base to the sugar (the glycosyl bond) to a C-C rather than an N-C bond (Figure 4). This modified base is now able to hydrogen bond with more than just its normal base pair, which is thought to have implications in overall RNA structures. The "a" and "d" notation in Fig. 4 denotes atoms in the structure that participate in the hydrogen bonds that form a normal base pair. The "a" points to an "acceptor" of a hydrogen from another atom (an adenine base in the case of normal base pairing). The "d" stands for "donor": an atom that donates, or shares, its hydrogen atom with an the adenine base that uridine is base pairing with. Notice that there is one more "donor" in the structure of pseudouridine than there is in uridine. Since RNA can form much more complex structures than just the basic double helix of DNA, this additional bonding ability is likely crucial to formation of mature RNA structure, like in ribosomes. In addition to forming the structure of the ribosome, these pseudouridine bases have also been found to contribute to the ribosome's interactions with mRNA and tRNA during the translation process. Figure 5 gives an example of pseudouridine's role in the complex structure of an rRNA. The filled-in circles and squares represent clusters of pseudouridine bases. The ladder-like portions of the figure represent normally base-paired regions of the rRNA. It is visible that sites of pseudouridylation participate in more than base pairing alone. The clusters of pseudouridines are positioned in proximity to many "ladder-like" segments of rRNA. The dotted diagonal line in the figure shows a 3-dimensional folding of the rRNA, that very likely occurs due to interactions involving nearby pseudouridines.

Figure 5. Pseudouridine clusters, represented by filled-in 

shapes, are involved in complex rRNA structure; 

adapted from Charette & Gray (2000)

Changes to molecules like the nitrogenous bases are performed by enzymes within the cell. Pseudouridine synthase is the enzyme responsible for pseudouridylation. However, this enzyme can't bind its target RNA on its own, it needs help from another molecule to recognize its target RNA. Guess who helps! Another RNA called snoRNA. Pseudouridine synthase is one of the four proteins that bind to snoRNA to form the snoRNA ribonucleoprotein. SnoRNA is able to act as a guide for the synthase, facilitating the enzyme's binding to its target RNA to modify the target's uracil bases. SnoRNA helps the enzyme identify the specific places that a given rRNA needs to be pseudouridylated. Without its RNA guide, the enzyme would be lost! 

A ribonucleoprotein (RNP) is a molecular complex made of both ribonucleic acid (RNA) and protein. Small nucleolar RNAs (snoRNAs) are RNAs that are transcribed and function as the RNA product; they do not carry messages to the ribosome like mRNA. SnoRNAs bind to pseudouridine synthase and other proteins to form the snoRNP. There are several known families, or types, of snoRNA that are classified into different groups based on commonalities in structure of the snoRNA and the proteins it binds with. "H/ACA" box snoRNAs are one family. Their name comes from the RNA structure that is conserved across all members of the H/ACA box family. The H/ACA box is a sequence of bases in the snoRNA structure that guides the pseudouridine synthase to the location of target RNA to be modified. 

Figure 6. H/ACA RNP structural interaction adapted from
Wu & Feigon (2007)

Figure 6 shows the snoRNP of the H/ACA box family. The figure illustrates the interface between the RNA and protein portions of the RNP. The RNA is shown in "stick and ribbon" form, allowing the nucleotide's structures to be seen while simplifying the backbone.  Different portions of the snoRNA's complex structure are colored green, purple, magenta, orange, and gray. These colored sections represent different structural subshapes the snoRNA forms to create its final and functional conformation. The protein component is show in "cartoon" light green and pink. The light green protein, labelled β, is one of several proteins that binds the snoRNA, but does not act on target mRNA as as enzyme. The pink protein, labeled α, is the pseudouridine synthase (the enzyme that modifies target uridine bases). The figure clearly shows the close interaction between the snoRNA and the pseudouridine synthase, enabling snoRNA to guide the enzyme to the correct uridine base on its target mRNA (not shown in figure).

The snoRNP is just one example of the complex world of RNAs, their involvement in enzymatic catalysis, and their diverse functions. SnoRNA interacts with an enzyme to help rRNA reach maturation. Nothing acts alone. Without pseudouridine synthase, rRNA would never fold into ribosomal subunits. And without snoRNA, pseudouridine synthase would struggle to find its target rRNA, and would likely pseudouridylate at the wrong uridine bases! The successful snoRNP keeps rRNA on track to its future as a ribosome, a molecular machine that the cell's vitality depends on limitlessly. 

Sources:
Charette, M.; Gray, M. W. Pseudouridine in RNA: What, Where, How, and Why IUBMB Life 2000, 49, 341.
Wu, H.; Feigon, J. H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification. PNAS 2007, 104, 6655.